Over the past 25 years, cancer immunotherapy has undergone tremendous innovation and progress with developments running parallel to advances in basic immunology, molecular genetics and gene therapy. Many of these treatments which have included potent cytokine infusions, the adoptive transfer of activated lymphocytes, and a wide array of cancer vaccines, have been largely applied to patients with metastatic melanoma. These treatments, however, have been consistently hampered by poor immunogenicity, limited immune durability, immune tolerance and tumor escape. To improve upon existing treatments and to help answer basic questions about mechanisms that fundamentally inhibit immune responses to cancer, we sought to develop a novel translational model of cancer immunotherapy. Supported by our preliminary studies, we focused our experimental efforts on further understanding the effects of antigen expression on lymphocyte selection, immune activation, autoimmunity, and cancer treatment. To investigate these issues, we designed a melanoma-based model composed of four critical components: 1) the use of two different experimental mice, one expressing a human class II allele (HLA-DR4), the other knocked-out in the expression of a melanocyte-differentiation antigen called tyrosinase-related-protein-1 (TRP-1); 2) the study of a crucial subset of T-cells (T-helper cells or CD4+ T-cells) which react against TRP-1; 3) the use of lentiviral technology to deliver to hematopoetic stem cells (HPSC) a CD4+ T-cell receptor (TCR) which reacts against TRP-1; and 4) the transplantation and immune reconfiguration with gene-modified HPSC. To achieve these ends we have broken down the overall goal into three specific aims: 1) Does gene-modified HPSC transplantation lead to competent T-cell development and function and can that functionality be further amplified with supplemental IL-7, vaccination, or by early antigen-driven homeostatic proliferation? 2) Does endogenous TRP-1 expression have a direct impact on immunoselection and immune tolerance following gene-modified HPSC transplantation? 3) What are the autoimmune, anti-tumor effects, and mechanisms-of-action of TRP-1-specific CD4+ T-cells following gene-modified HPSC transplantation? We believe this research proposal builds upon existing treatments and technologies in cancer immunotherapy. What makes this model unique however, is both the role of antigen to control the composition of the T-cell repertoire and the critical position that CD4+ T-cells play in global immune activation and anti-tumor activity. We believe that experimental approaches, which facilitate the long-term engraftment, expansion and activation of highly reactive CD4+ T-cells, ultimately produce more effective cancer immunotherapies. PUBLIC HEALTH RELEVANCE: This research proposal builds upon existing technologies and treatments in gene-therapy and immunotherapy for patients with cancer and metastatic melanoma in particular. Here we demonstrate the development of a model in which we successfully reconfigure the immune system with a specific high-frequency white blood cell that is capable of recognizing a key protein found on the surface of melanoma. We then propose experiments designed to better understand the therapeutic implications of this model, improve its overall effectiveness, and study its mechanisms-of-action.